Large insert DNA libraries are essential for the molecular analysis of the human genome and other genomes important to our well-being. Comparative genomics reveals new information about metabolic pathways, cancer, horizontal gene transfer, evolution, protein families, and the genetic repertoire of numerous species. It has also greatly increased the demand for additional genomic BAC libraries. Construction of these libraries is time consuming, costly and challenging. The long-term objectives of this proposal are to facilitate cloning the current gaps in the human genome and reduce the time required for constructing large BAC libraries to a few weeks from several months. The quality of these libraries will surpass current standards for bias, randomness, fidelity, and low contamination. Specific aims include the development of new BAC cloning tools, strains, and methods that circumvent traditional bottlenecks and substantially improve the molecular cloning and transformation efficiency of high molecular weight genomic DNA. A new E. coli transformation system could improve large DNA uptake 100-fold and increase average insert size to above 300 kb. Other technical innovations include a simple method for randomly shearing high molecular weight DNA, a zero background cloning system, and a transcription-free cloning system that eliminates several forms of cloning bias. PROPOSED COMMERCIAL APPLICATIONS: Commercial products and services include: a new E. coli transformation system with exceptionally high DNA uptake efficiency, a zero background, transcription-free vector for unbiased cloning of recalcitrant genes and gneomes, a BAC library construction kt, a BAC DNA affinity purification kit, a vector pop-out to reduce re-sequencing of the plasmid backbone, and an economical BAC library construction service.